2B) after cleavage of the C-terminal propeptide (amino acids 103 to 130). Open 1,2-Dipalmitoyl-sn-glycerol 3-phosphate in a separate window FIG 2 cDNA expression library screening to identify the syncytin-A partner of fusion. fusion assay with the screening of a mouse cDNA library, we succeeded in identifying the glycosylphosphatidylinositol (GPI)-anchored membrane protein lymphocyte antigen 6E (Ly6e) as a candidate receptor for SynA. Transfection of cells with the cloned receptor led to their fusion to cells expressing SynA, with no cross-reactive fusion activity with SynB. Knocking down Ly6e greatly reduced SynA-induced cell fusion, thus suggesting that Ly6e is the sole receptor for SynA fusion assay that enables the high-throughput screening of normalized cDNA libraries, we identified the long-sought receptor for syncytin-A (SynA), a mouse syncytin responsible for syncytiotrophoblast formation at the maternofetal interface of the mouse placenta. This protein, Ly6e (lymphocyte antigen 6E), is a GPI-anchored 1,2-Dipalmitoyl-sn-glycerol 3-phosphate membrane protein, and small interfering RNA (siRNA) experiments targeting its deletion as well as a decoy assay using a recombinant soluble receptor show that Ly6e is the necessary and sufficient partner of SynA. Its profile of expression is consistent with a role in both ancestral endogenization of a SynA founder retrovirus and present-day placenta formation. This study provides a powerful general method to identify genes involved in cell-cell fusion processes. genes are envelope (genes have been found in all major clades of placental mammals and result from independent captures of genes in each lineage where they have been identified (4,C11). The genes display conserved characteristics from genes but also lineage-specific differences which correlate with placental structure differences. The structure of the mouse placenta is unique among placental mammals: at variance with all other described placentae, the fetomaternal interface comprises two syncytiotrophoblast layers (ST-I and ST-II) instead of one, as observed in humans and all other hemochorial placentae (12). Each of these syncytiotrophoblast layers expresses a different syncytin: syncytin-A (SynA) 1,2-Dipalmitoyl-sn-glycerol 3-phosphate for ST-I 1,2-Dipalmitoyl-sn-glycerol 3-phosphate and syncytin-B (SynB) for ST-II (13). Both syncytins have been demonstrated to be required for placenta development, with altered structures of the fetomaternal interface in knockout (KO) mice (14, 15). SynA KO mice display the most severe phenotype, resulting in death of embryos at midgestation. By analogy with other retroviral Envs, syncytin-induced cell-cell fusion is thought to be mediated by the interaction of the syncytin with a specific membrane receptor expressed on neighboring cells (16). In this model, syncytin-mediated cell-cell fusion is initiated when the surface subunit (SU) of the syncytin glycoprotein binds to Mouse monoclonal to ESR1 a specific receptor expressed on the surface of a neighboring cell. Attachment induces a series of conformational changes of the SU and transmembrane (TM) subunits of the syncytin, which results in the fusion of the plasma membranes. It was previously demonstrated that SynA and -B overexpression induces cell-cell fusion (10). As SynA and -B overexpression does not induce fusion in the same cell lines, it was hypothesized that their cellular receptors had to be distinct (10). Among the 11 mammalian syncytins characterized so far, the receptors for human syncytin-1 and -2 (human ASCT2/SLC1A5/RDR and human MFSD2/NLS1, respectively) and rabbit syncytin-Ory1 (rabbit ASCT2) have been identified by using pseudotyping assays (5, 17, 18). Although SynA and -B were first identified more than 10 years ago, the corresponding receptors 1,2-Dipalmitoyl-sn-glycerol 3-phosphate have not been identified yet. This was mainly due to the difficulty in assessing interactions between membrane proteins, the impossibility of generating functional pseudotypes with SynA or -B (data not shown), and the lack of appropriate assays to screen cDNA libraries for cell-cell fusion is then investigated, as well as its ability to interact with SynA and its tissue expression profile. RESULTS Development of a cell fusion-based screening method. Given that SynA does not generate functional pseudotypes (probably due to improper incorporation into viral particles), we searched for a cell-cell fusion assay which would be simple and sensitive enough to screen a cDNA library. -Complementation of -galactosidase was previously used to detect HIV envelope glycoprotein-mediated cell fusion (19). We first checked that -complementation was.